Explore the vast range of titles available.

is a major pathogen for swine and human. Here we aimed to know the rates of antimicrobial resistance (AMR) in invasive isolates recovered along Spain between 2016 - 2021 and elucidate their genetic origin. Antibiotic susceptibility testing was performed for 116 isolates of different genetic backgrounds and geographic origins against 18 antibiotics of 9 families. The association between AMR and genotypes and the origin of the isolates were statistically analyzed using Pearson´s chi-square test and the likelihood ratio. The antimicrobial resistant genes were identified by whole genome sequencing analysis and PCR screenings. High AMR rates (>80%) were detected for tetracyclines, spectinomycin, lincosamides, and marbofloxacin, medium (20-40%) for sulphonamides/trimethoprim, tiamulin, penicillin G, and enrofloxacin, and low (< 20%) for florfenicol, and four additional β-lactams. The occurrence of multidrug resistance was observed in 90% of isolates. For certain antibiotics (penicillin G, enrofloxacin, marbofloxacin, tilmicosin, and erythromycin), AMR was significantly associated with particular sequence types (STs), geographic regions, age of pigs, and time course. Whole genome sequencing comparisons and PCR screenings identified 23 AMR genes, of which 19 were previously reported in ( (3')-IIIa, , aac(6')-Ie- (2'')-Ia, (B), (A/E), (D), (C), (B), (E), (F), (M), (O), (O/W/32/O), (W)), and 4 were novel ( (2'')-IIIa, (47), (T)). These AMR genes explained the AMR to spectinomycin, macrolides, lincosamides, tiamulin, and tetracyclines. Several genes were located on mobile genetic elements which showed a variable organization and composition. As AMR gene homologs were identified in many human and animal pathogens, the resistome of has a different phylogenetic origin. Moreover, AMR to penicillin G, fluoroquinolones, and trimethoprim related to mutations in genes coding for target enzymes ( , , and . Bioinformatic analysis estimated traits of recombination on target genes, also indicative of gene transfer events. Our work evidences that is a major contributor to AMR dissemination across veterinary and human pathogens. Therefore, control of AMR in should be considered from a One Health approach in regions with high pig production to properly tackle the issue of antimicrobial drug resistance.

The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

Few studies have focused on assessing Salmonella infection in the nursery and its role in further pig production periods. Mesenteric lymph nodes, intestinal content, and meat juice from 389 6-week-old male piglets intended for human consumption from five breeding farms and 191 pooled floor fecal samples from gilt development units (GDU) from the same farms were analyzed to estimate and characterize (by pulsed-field gel electrophoresis and antimicrobial resistance analyses) Salmonella infection. The prevalence of infection and shedding among piglets was 36.5% and 37.3%, respectively, shedding being significantly associated with infection (Odds Ratio = 12.7; CI 7.3–22.0). Salmonella Rissen; S. 4,[5],12:i:-; and S. Derby were the most common serotypes. A low level of Salmonella-specific maternal antibodies at the beginning of the nursery period suggested it was a period of high risk of infection. Resistance to 3rd- and 4th-generation cephalosporins was detected in piglet isolates although the piglets never received antibiotics, indicating they could be vectors of antimicrobial resistance. The same Salmonella clones were detected in piglet and GDU isolates, suggesting that infected piglets play a significant role in the infection of gilts and consequently of finishing pigs in the case of production farms. The control of Salmonella infection in nursery piglets may decrease the risk of abattoir and carcass contamination.

Streptococcus suis is a zoonotic pathogen that causes a major health problem in the pig production industry worldwide. Spain is one of the largest pig producers in the world. This work aimed to investigate the genetic and phenotypic features of invasive S. suis isolates recovered in Spain. A panel of 156 clinical isolates recovered from 13 Autonomous Communities, representing the major pig producers, were analysed. MLST and serotyping analysis revealed that most isolates (61.6%) were assigned to ST1 (26.3%), ST123 (18.6%), ST29 (9.6%), and ST3 (7.1%). Interestingly, 34 new STs were identified, indicating the emergence of novel genetic lineages. Serotypes 9 (27.6%) and 1 (21.8%) prevailed, followed by serotypes 7 (12.8%) and 2 (12.2%). Analysis of 13 virulence-associated genes showed significant associations between ST, serotype, virulence patterns, and clinical features, evidencing particular virulence traits associated with genetic clusters. The pangenome was generated, and the core genome was distributed in 7 Bayesian groups where each group included a variable set of over- and under-represented genes of different categories. The study provides comprehensive data and knowledge to improve the design of new vaccines, antimicrobial treatments, and bacterial typing approaches.

The live attenuated Brucella melitensis Rev.1 strain is considered the best vaccine available for the prophylaxis of brucellosis in sheep caused by either B. melitensis or Brucella ovis. However, its application stimulates antibody responses in vaccinated animals indistinguishable by the current conventional serological tests from those observed in infected animals. The periplasmic protein BP26 and the outer membrane protein (OMP) Omp31 are immunodominant antigens in the serological responses of B. melitensis and B. ovis infected sheep, respectively. Accordingly, vaccine strain Rev.1 single and double deletion mutants of the bp26 and omp31 genes were developed, based on the principle that the use of such mutants as vaccines in association with diagnostic tests based on BP26 and Omp31 antigens would allow the serological differentiation between infected and vaccinated animals. The deletion mutants obtained were indistinguishable from the parental Rev.1 strain by conventional bacteriological and typing tests. The expression of their major surface antigens, as determined by reactivity with specific monoclonal antibodies (MAbs), remained unaffected, i.e. smooth-lipopolysaccharide (S-LPS) and OMPs besides in the expression of the antigens whose respective genes were deleted. The bp26 and omp31 deletions did not modify the kinetics of splenic infection nor the residual virulence of Rev.1 in the BALB/c mouse model. Vaccination of BALB/c mice with the deletion mutants conferred significant protective immunity against B. melitensis strain H38 or B. ovis strain PA challenges, to the same extent as that induced by parental Rev.1 strain. Thus, these Rev.1 bp26 or omp31 deletion mutants are promising vaccine candidates against B. melitensis and B. ovis infections and will be further evaluated in sheep.

Background The role of wildlife as a brucellosis reservoir for humans and domestic livestock remains to be properly established. The aim of this work was to determine the aetiology, apparent prevalence, spatial distribution and risk factors for brucellosis transmission in several Iberian wild ungulates. Methods A multi-species indirect immunosorbent assay (iELISA) using Brucella S-LPS antigen was developed. In several regions having brucellosis in livestock, individual serum samples were taken between 1999 and 2009 from 2,579 wild bovids, 6,448 wild cervids and4,454 Eurasian wild boar ( Sus scrofa ), and tested to assess brucellosis apparent prevalence. Strains isolated from wild boar were characterized to identify the presence of markers shared with the strains isolated from domestic pigs. Results Mean apparent prevalence below 0.5% was identified in chamois ( Rupicapra pyrenaica ), Iberian wild goat ( Capra pyrenaica ), and red deer ( Cervus elaphus ). Roe deer ( Capreolus capreolus ), fallow deer ( Dama dama ), mouflon ( Ovis aries ) and Barbary sheep ( Ammotragus lervia ) tested were seronegative. Only one red deer and one Iberian wild goat resulted positive in culture, isolating B. abortus biovar 1 and B. melitensis biovar 1, respectively. Apparent prevalence in wild boar ranged from 25% to 46% in the different regions studied, with the highest figures detected in South-Central Spain. The probability of wild boar being positive in the iELISA was also affected by age, age-by-sex interaction, sampling month, and the density of outdoor domestic pigs. A total of 104 bacterial isolates were obtained from wild boar, being all identified as B. suis biovar 2. DNA polymorphisms were similar to those found in domestic pigs. Conclusions In conclusion, brucellosis in wild boar is widespread in the Iberian Peninsula, thus representing an important threat for domestic pigs. By contrast, wild ruminants were not identified as a significant brucellosis reservoir for livestock.

Due to the important drawbacks of the Brucella melitensis Rev 1 vaccine, a safer vaccine based on an outer membrane complex from Brucella ovis encapsulated in poly-ɛ-caprolactone (PEC) microparticles (MP) was developed and tested in rams. Homogeneous batches of microparticles were prepared by a new double emulsion solvent evaporation method called “Total Recirculation One-Machine System” (TROMS). Such microparticles presented a mean diameter of 2 μm and displayed an antigen loading of about 13 μg HS per mg of microparticles. Subcutaneous vaccination of rams with 800 μg HS (hot saline antigenic extract of B. ovis) in PEC microparticles induced an adequate serological response against B. ovis antigens and conferred similar protection against challenge with B. ovis to that induced by the living attenuated B. melitensis Rev 1 reference vaccine. By contrast, lower doses (80 μg) of HS-PEC evoked reduced serological responses against B. ovis antigens and did not induce significant protection. The revaccination with 800 μg of HS-PEC increased the intensity and duration of the serological response against B. ovis antigens but did not improve the protection conferred by the single vaccination. Sample sera taken from any of the animals immunized with Rev 1 were seropositive in both Rose Bengal and the Complement Fixation tests (RBT, CFT) used for the diagnosis of smooth Brucella infections. By contrast, no positive reactors in both tests were recorded in the animals vaccinated with HS-PEC, being this a target objective of this study. HS-PEC microparticles can be used as a safe vaccine against brucellosis in rams, but further studies using higher doses of antigens are necessary to exploit their full potential for the prophylaxis of brucellosis in sheep.

Classical brucellosis vaccines induce antibodies to the O-polysaccharide section of the lipopolysaccharide that interfere in serodiagnosis. Brucella rough (R) mutants lack the O-polysaccharide but their usefulness as vaccines is controversial. Here, Brucella melitensis R mutants in all main lipopolysaccharide biosynthetic pathways were evaluated in sheep in comparison with the reference B. melitensis Rev 1 vaccine. In a first experiment, these mutants were tested for ability to induce anti-O-polysaccharide antibodies, persistence and spread through target organs, and innocuousness. Using the data obtained and those of genetic studies, three candidates were selected and tested for efficacy as vaccines against a challenge infecting 100% of unvaccinated ewes. Protection by R vaccines was 54% or less whereas Rev 1 afforded 100% protection. One-third of R mutant vaccinated ewes became positive in an enzyme-linked immunosorbent assay with smooth lipopolysaccharide due to the core epitopes remaining in the mutated lipopolysaccharide. We conclude that R vaccines interfere in lipopolysaccharide immunosorbent assays and are less effective than Rev 1 against B. melitensis infection of sheep.

The assessment of the genetic stability is one of the essential elements to guarantee the biological quality of live anti-bacteria vaccines. Live attenuated Brucella melitensis Rev 1 is the most effective vaccine against brucellosis in small ruminants. Thirty-six B. melitensis Rev 1 vaccine strains isolated from human or animal sources from different geographic regions, from different commercial batches or laboratory collections were typed by the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) recently described for Brucella spp. Our results demonstrated that B. melitensis Rev 1 group as assayed by MLVA is genetically very homogeneous. We believe that MLVA methodology could be an essential assay to guarantee the quality and stability of live anti-bacterial vaccines being produced worldwide and can be included as in vitro control.